猴免疫缺陷病毒SIV核心蛋白基因在大肠杆菌中表达的研究

应用多聚酶链反应（ＰＣＲ）,直接从ＳＩＶ感染的猴艾滋病（ＳＡＩＤＳ）模型猴的外周血淋巴细胞总ＤＮＡ中扩增出７６７ｂｐ的ＳＩＶ核心蛋白Ｐ２７基因片段。扩增产物经ＥｃｏＲＩ及ＳａｌＩ双酶切后,克隆入相同酶切的表达质粒ｐＢＶ２２０中,获得含ＳＩＶ核心蛋白基因片段的重组质粒ｐＢＶＳＧ,并进行ＤＮＡ序列分析。用该重组质粒转化大肠杆菌ＤＨ５ａ经筛选、增殖及４２℃温度诱导,ＳＤＳ－ＰＡＧＥ表明外源基因表达蛋白含量占菌体总蛋白１４．５％,Ｗｅｓｔｅｒｎ－ｂｌｏｔ证实表达产物能被ＳＩＶＰ２７单克隆抗体及ＳＡＩＤＳ模型猴血清中特异性抗体识别。     

牛泡沫病毒（BSV）3026毒株的分离及分子生物学鉴定

从一头牛免疫缺陷病毒（ＢＩＶ）检测阳性３０２６号牛外周血中,分离到一株病毒,即３０２６病毒株。体外细胞增减和反转录分析证明,此病毒是一反转录病毒。可胎牛肺细胞中引起典型的泡沫样病变,形成合胞体,ＰＣＲ扩增和Ｓｏｕｔｈｅｍ杂交显示,此病毒的ＣＤＮＡ和ＰＣＲ产的均可与牛泡沫病毒（ＢＳＶ）阳性对照的ＨｉｒｔＤＮＡ杂交。３‘ＬＴＲ上游一段３３０ｂｐ的ＰＣＲ产物序理分析表明,３０２６毒株与ＢＳＶ阳性对照相比     

应用RT—PCR检测流产胎儿组织中猪繁殖与呼吸综合征病毒

根据猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）美洲型膜蛋白和核衣壳蛋白基因序列,设计了一对含有ＥｃｏＲＩ和ＢａｍＨＩ酶切位点的引物,用ＲＴ－ＰＣＲ对四个流产猪场的病料进行了检测,扩增出约９１８ｂｐ的基因片段。通过病毒分离、酶切鉴定和序列分析证实为ＰＲＲＳＶ感染。结果说明应用所设计的引物进行ＲＴ－ＰＣＲ快速检测ＰＲＲＳ是可行的,为我国快速特异诊为ＰＲＲＳ和ＰＲＲＳＶ强毒株的深入研究奠定了基础。     

PCR方法检测我国脊髓灰质炎病毒Ⅰ型野毒株的研究

用聚合酶链反应试验（ＰＣＲ）检测我国脊髓灰质炎病毒Ⅰ型（ＰＶＩ）野毒株,仅需５μｌＰＶＩ细胞培养液,方法简便、快速、敏感、特异,用本法对我国１４个省市７９份ＰＶＩ分离株的检测结果,能与检定ｓａｂｉｎⅠ相关株的ＳＩ／ＰＣＲ法的检测结果相印证,与其中３１份核苷酸测序判断为野毒株的结果相一致。其检测阳性率为８４．４％,基本能检测我国流行过的５个ＰＶＩ基因型野毒株。另外,从检测结果看,除北京市未发现野毒株外,其它１３个省区都有野毒株流行,表明当前我国消灭脊灰的任务还很重。     

原位多聚酶链反应在人巨细胞病毒诊断中的应用

逝年来人巨细胞病毒（ＨＣＭＶ）感染率高,检测方法进展人,有巨细胞包涵体细胞（ＣＣＩＣ）检测、病毒分离、免疫检测、原位杂交（ＩＳＨ）、多聚酶链反应（ＰＣＲ）等法。目前有人把ＩＳＨ与ＰＣＲ结合创立了原位多聚酶链反应法（ＩＳＰＣＲ）,此法更准确、更敏感特异,更有地ＨＣＭＶ感染的早期快速诊断。本文就ＩＳＰＣＲ的原理、程序、特征主临床应用作一综述。     

犬细小病毒中国内蒙株VP2基因克隆及序列分析

从我国内蒙古地区流行的犬细小病毒病病犬的肠溶物中分离提纯犬细小病毒（ＣＰＶ）。提取病毒基因组ＤＮＡ,并以此ＤＮＡ为模板,采用人工合成的引物进行ＰＣＲ扩增,ＰＣＲ产物经ＢａｍＨＩ、ＳａｃＩ双酶切后,克隆于ｐＵＣ１９质粒的ＢａｍＨＩ／ＳａｃＩ位点。重组质粒ｐＵＣＶＰ２经ＰＣＲ鉴定、限制酶切分析和序列分析,结果表明：获得了犬细小病毒内蒙株（ＣＰＶ－ＩＭ）ＶＰ２基因的全长克隆,ＶＰ２基因全长１７５５ｎｔ,     

应用套式PCR对恶性卡他热病毒在不同动物体内变异的研究

应用本实验建立的三组套式ＰＣＲ（ＰＣＲ１、２、３）和一组以前报道的套式ＰＣＲ（ＰＣＲ４）,对５９份外周血淋巴细胞（ＰＢＬ）ＤＮＡ样品进行了恶性卡他热病毒（Ｍａｌｉｇｎａｎｔｃａｔａｒｒｈａｌｆｅｖｅｒｖｉｒｕｓ,ＭＣＦＶ）核酸序列的检测。这些样品来自５１只羊,以及与羊接触而发病的６头牛和２只鹿。除ＰＣＲ４外,其它三组ＰＣＲ都能扩增现有４个角马型ＭＣＦＶ分离株。有６只羊在４组ＰＣＲ中都呈阴性,其余５３份样品经ＰＣＲ４检测均呈阳性。ＰＣＲ１只能从４５只羊体检出ＭＣＦＶＤＮＡ,未能从牛和鹿体检出病毒ＤＮＡ。ＰＣＲ２检测的所有样品均呈阴性。在ＰＣＲ３扩增中,除２头牛外,其它５１份样品均呈阳性。通过Ｓｏｕｔｈｅｒｎ杂交和限制性酶切分析,对ＰＣＲ１－４产物的特异性进行了鉴定。此外,敏感性实验表明,四组ＰＣＲ的差异也不明显。因此,本实验结果说明ＭＣＦＶ基因组在不同种动物之间发生了变异,羊体内的变异株可能是导致其它反刍动物发病的病原     

RT—nested PCR检测肾综合征出血热患者血清病毒核酸

采用异硫氰酸胍－酚－氯仿（ＡＧＰＣ）一步法提取病毒ＲＮＡ,并依据肾综合征出血热病毒（ＨＦＲＳＶ）核蛋白（ＮＰ）编码基因保守区核苷酸序列合成两对巢式引物,建立了逆转录巢式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄＰＣＲ）检测ＨＦＲＳＶＲＮＡ方法,应用此法对ＨＦＲＳＶ感染的ＶｅｒｏＥ６细胞培养液及ＨＦＲＳ患者血清中的病毒ＲＮＡ进行检测。结果显示,感染细胞培养液及３５例ＨＦＲＳ患者血清均为阳性,正常的ＶｅｒｏＥ６     

直接逆转录原位PCR检测实验性汉坦病毒感染乳鼠脑组织中病毒RNA及其与原位分子杂交的比较

出生后２～３ｄ的昆明种乳鼠,经腹腔接种１００个半数致死量的陈株汉坦病毒,每只０．０５ｍｌ,于接种后１、２、４、６、８、１０、１２、１４ｄ处死动物,每个时间点３～６只不等,取其脑组织固定于４％的多聚甲醛中,石蜡包埋制备５μｍ的连续组织切片,每时间点取１～２例组织切片,用逆转录原位ＰＣＲ（ＲＴ－ＩＳＰＣＲ）方法检测组织中病毒Ｓ片段ＲＮＡ,组织脱蜡后,经ＤＮａｓｅ、蛋白酶Ｋ、消化等予处理,用汉坦病毒ＲＮＡＳ片段特异的一对引物,在组织切片上进行病毒ＲＮＡ的逆转录和ＰＣＲ过程,直接将ｄｉｇｏｘｉｇｅｎｉｎ－１１－ｄＵＴＰ掺入到扩增产物中,经过３０个ＰＣＲ循环后,用碱性磷酸酶标记的抗ｄｉｇｏｘｉｇｅｎｉｎ抗体免疫组化检测扩增产物,连续组织切片用ｄｉｇｏｘｉｇｅｎｉｎ标记的汉坦病毒Ｍ片段Ｇ２编码区ＲＴ－ＰＣＲ扩增产物的和Ｓ片段特异性探针进行原位分子杂交并与ＲＴ－ＩＳＰＣＲ结果进行比较,另外应用免疫组化检测该基因表达产物病毒核抗原（ＮＰ）,结果,ＲＴ－ＩＳＰＣＲ在病毒感染１ｄ的乳鼠脑组织中检测到病毒ＲＮＡ扩增产物,扩增产物定位于神经细胞胞浆内,而原位分子杂交和免疫组化检测阴性。在病毒感染２ｄ及２ｄ以后的乳鼠脑组织中ＲＴ－Ｉ     

应用循环逆转录PCR技术检测丙型肝炎病毒RNA

循环逆转录（ｃｉｒｃｕｌａｔｏｒｙ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ,ＣＲＴ）是线性增长逆转录ｃＤＮＡ产量的一种新技术。为了将该技术用于检测ＨＣＶ ＲＮＡ,通过改变ＣＲＴ的循环次数,结合竞争ＰＣＲ,作出标准曲线。采用１６次ＣＲＴ加３４次循环ＰＣＲ检测了１３６例ＨＣＶ ＥＬＩＳＡ阳性、５４例ＨＣＶ ＥＬＩＳＡ阴性和１０８例临床可疑病人全血标本,并与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）和巢式ＰＣＲ（     

PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies

We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (10(8) cells) plus 8 ml of whole blood from protected animals to na?ve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.     

PCR检测伪狂犬病病毒DNA

 根据伪狂犬病病毒 (PRV)gB基因的序列 ,设计并合成了一对引物 ,以闽A株细胞培养毒为模板 ,筛选最佳反应条件 ,建立了检测PRV的PCR方法 应用该方法对Fb、Bartha、BJ、GD、V2F4、S、S3、SR、Buk、Shope、Norden、MinkⅢ、HB、F8、F9、F12等毒株的细胞培养液进行基因扩增 ,均获得了分子量为 2 81bp的特异性目的DNA片段 ,而对Vero细胞与FMDV、SVDV、HCV、PRRSV、JEV、PPV等病毒进行检测 ,结果均为阴性 ,没有出现交叉反应 对PRV毒株扩增的产物测序 ,结果序列与文献报道一致 ,证明PCR扩增产物和方法的特异性 对 1994～ 2 0 0 0年期间送检的临床样品和保存的PRV毒种 ,用病毒分离、双抗体夹心ELISA和PCR等 3种方法进行检测 ,结果前 2种方法检测为阳性的 ,PCR检测均为阳性 ;PCR检测为阴性 ,前 2种方法检测也为阴性 ;可是 ,前 2种方法检测为阴性的 ,PCR却检测出部分阳性 ;经x2 检验 ,证明PCR检出率明显高于前 2种方法的检出率 对PRV闽A株细胞毒提取物DNA进行检测 ,其最低检出量为 15 8pg 对 1999～ 2 0 0 0年期间广东、福建、海南等省的 31个大中型猪场送检的 191份病料进行检测 .结果病料阳性率为 2 6 2 % ( 50 191) ,猪场阳性率为 71% ( 2 2 31) 实验结果表明 ,所建立的PCR技术可用于伪狂犬病的快速诊断     

Simian immunodeficiency virus (SIV) gag DNA-vaccinated rhesus monkeys develop secondary cytotoxic T-lymphocyte responses and control viral replication after pathogenic SIV infection

The potential contribution of a plasmid DNA construct to vaccine-elicited protective immunity was explored in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Making use of soluble major histocompatibility class I/peptide tetramers and peptide-specific killing assays to monitor CD8(+) T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gag DNA vaccine construct was shown to elicit a high-frequency SIV-specific cytotoxic T-lymphocyte (CTL) response. This CTL response was demonstrable in both peripheral blood and lymph node lymphocytes. Following an intravenous challenge with the highly pathogenic viral isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were comparable in the experimentally and control-vaccinated monkeys during the period of primary infection, the gag plasmid DNA-vaccinated monkeys demonstrated better containment of viral replication by 50 days following SIV challenge. These findings indicate that a plasmid DNA vaccine can elicit SIV-specific CTL responses in rhesus monkeys, and this vaccine-elicited immunity can facilitate the generation of secondary CTL responses and control of viral replication following a pathogenic SIV challenge. These observations suggest that plasmid DNA may prove a useful component of a human immunodeficiency virus type 1 vaccine.     

Presence of caprine arthritis-encephalitis virus (CAEV) proviral DNA in genital tract tissues of superovulated dairy goat does

Transmission of caprine arthritis-encephalitis virus (CAEV) is not completely understood and the vertical route of infection from the goat to the embryo or to the fetus needs to be investigated. This route of infection involves the presence of CAEV in the genital tract tissues. Prior studies have detected CAEV-infected cells in genital secretions and in flushing media recovered during embryo collection from infected goats. To specify the origin of these cells, we conducted a double-nested polymerase chain reaction (PCR) test on embryo flushing media and on mammary gland, mammary lymph node, synovial membrane, pelvic lymph node, uterus and oviduct tissues from 25 CAEV-infected (blood PCR positive) embryo donor goats for the presence of CAEV proviral DNA.The presence of proviral DNA was found in 22 of 25 mammary gland samples, 14 of 25 uterus samples, and in 16 of 25 oviduct samples. Nineteen of 25 goats had at least one positive genital tract sample. Flushing media from 11 goats were PCR positive. All goats with positive-flushing media were oviduct positive. Of this group of does, except for 1 of the 11, infection of flushing media correlated with infection of almost all the other tissues examined. The frequency of positive tissues for flushing media-positive goats (61/66; 92%) was significantly higher than that for flushing media-negative goats (50/84; 60%) (P<0.01).This study demonstrated the presence of CAEV-infected cells in the goat genital tract. The presence of CAEV-infected cells in the uterus and oviducts suggests potential for vertical transmission of CAEV from doe to embryo or fetus.     

Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses

The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20(+) B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection.     

Infection of rhesus monkeys with topical instillation of simian immunodeficiency virus (SIV)B670 into the conjunctival sac

We tested the ability of SIV to cause local and systemic infection in three rhesus monkeys after topical instillation of cell-free virus into the conjunctival cul-de-sac. Conjunctivitis or other signs of infection were monitored after inoculation. Conjunctiva were swabbed for virus culture and biopsied for PCR. Changes in lymphocyte subsets, seroconversion, antigenemia, and virus isolation from PBL were assessed systemically postinoculation. Viral DNA was detected in conjunctival biopsy by PCR in one of three animals that later developed systemic infection. The other two animals remained uninfected. These data demonstrate that the conjunctiva is a route by which SIV (and perhaps HIV) may cause systemic infection.     

Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation

HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA) levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta) that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1–9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA) in the axillary lymph node (6.48±0.50) were significantly higher than in the genital tract tissues: testis (3.67±2.16; p<0.05), epididymis (3.08±1.19; p<0.0001), prostate (3.36±1.30; p<0.01), and seminal vesicle (2.67±1.50; p<0.0001). Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.     

Long-term follow-up study on SIV intestinal proviral load in rhesus macaques

After experimental infection with simian immunodeficiency virus (SIV), intestinal endoscopy proved to be an easily tolerated, minimal invasive procedure to obtain biopsies from the gastrointestinal tract of rhesus macaques during all stages of disease. As the GI tract is affected by many opportunistic infections and immunological impairment after SIV/human immunodeficiency virus (HIV) infection, knowledge on the proviral load is an important parameter for a better understanding of disease pathogenesis. In this paper, we describe the set-up and evaluation of a quantitative competitive polymerase chain reaction (PCR) and the quantification of SIV intestinal proviral load in a long-term follow-up study of eight rhesus monkeys ( Macaca mulatta ) after two different routes of virus inoculation. A SIV-specific signal could be detected as early as day 3 after infection. Of 143 biopsies from the follow-up study, 85.3% showed a positive PCR. DNA copy numbers ranged from 300 to 15,000 molecules per 100,000 cells. No significant influence of the inoculation route could be shown on either proviral load or survival time, but higher SIV proviral load was associated with a more rapid progression to disease. Therefore, the amount of proviral load in intestinal biopsies may be an important prognostic value for the further course of the disease.